Protein and lipid components of brain and lung tissue thromboplastin and platelet factor 3 have been isolated, extracted, and partially purified and characterized. The objectives of this proposal are: A) to fully characterize the components; B) To define molecular mechanisms of interaction required for the bioactivity; C) To determine cellular sites of protein synthesis, storage, and release; and D) To evaluate the mechanisms by which these insoluble procoagulants react with the soluble coagulant proteins in plasma. Studies involved in objective A include: 1) Further purification of the protein components by preparative polyacrylamide electrophoretic, isoelectric focus, and density gradient centrifugation methodologies; 2) Comparative characterization of the purified proteins with respect to; a) molecular weight; b) carbohydrate and amino acid composition and content; and c) primary structure; and 3) Further characterization of lipid components for a) fatty acid composition and b) composition of the neutral lipids by TLC and column chromatographic techniques. In objective B, single-compartment lipid vesicles from native and purified lipids will be recombined with the protein isolates to evaluate the lipid-protein recombination requirements for optimal activity restoration. Bioactivity will also serve as the basis for determining efficacy of recombination procedures. Biochemical characteristics of the recombined products will be compared to lipid vesicles and proteins alone. Similar studies using vesicles composed of purified lipids will also be performed. Protein isolates will be employed in immunologic studies of the cellular site of synthesis, storage and release. Monospecific antibodies will be developed and used in labelling and inhibitor studies to attain objective C. Objective D will be approached by monitoring the in vitro reactions between chemically defined procoagulant lipoprotein vesicles and purified coagulation proteins. Immunologic inhibition, radio labelling, and bioactivity will be used as tools to monitor complex formation.